Poly(levodopa)-modified β-glucan as a candidate for wound dressings

Carbohydrate Polymers 272 (2021) 118485

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Poly(levodopa)-modified β-glucan as a candidate for wound dressings
´ ska d, Anna Belcarz a, *
Anna Michalicha a, Agata Roguska b, Agata Przekora c, Barbara Budzyn
a

Chair and Department of Biochemistry and Biotechnology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland
Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
c
Independent Unit of Tissue Engineering and Regenerative Medicine, Chair of Biomedical Sciences, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland
d
Independent Laboratory of Behavioral Studies, Medical University of Lublin, Chodzki 4a, 20-093 Lublin, Poland
b

A R T I C L E I N F O

A B S T R A C T

Keywords:
β-Glucan
Poly(levodopa)-based modification
Fibroblasts
Danio rerio
Antibacterial activity

Levodopa (biological precursor of dopamine) is sometimes used instead of dopamine for synthesis of highly
adhesive polycatecholamine coatings on different materials. However, in comparison of polydopamine, little is
known about biological safety of poly(levodopa) coatings and their efficacy in binding of therapeutically active
substances. Therefore, thermally polymerized curdlan hydrogel was modified via two different modes using
levodopa instead of commonly used dopamine and then coupled with gentamicin – aminoglycoside antibiotic.
Physicochemical properties, biological safety and antibacterial potential of the hydrogels were evaluated. Poly
(levodopa) deposited on curdlan exhibited high stability in wide pH range and blood or plasma, were nontoxic in
zebrafish model and favored blood clot formation. Simultaneously, one of hydrogel modification modes allowed
to observe high gentamicin binding capacity, antibacterial activity, relatively high nontoxicity for fibroblasts and
was unfavorable for fibroblasts adhesion. Such modified poly(levodopa)-modified curdlan showed therefore high
potential as wound dressing biomaterial.

1. Introduction
Polydopamine (PDA) is a major pigment which occurs in natural
melanin (eumelanin) (Simon & Peles, 2010). It also mimics the
specialized adhesive foot protein (Mytilus edulis foot protein-5) in mus­
sels (Lee et al., 2007). Based on this phenomenon, the method of bio­
mimetic approach for the functionalization of a wide range of materials
has been developed and proposed in 2007 (Lee et al., 2007). Since then,
formation of PDA coating became very popular as a strategy of solid
substrate functionalization for a variety of technical, environmentprotecting and medical purposes. PDA coatings were used for example
for modification of graphene nanosheets (Wang et al., 2013; Xu et al.,
2010), Fe3O4 nanoparticles (for drug delivery, for catalyst support, ad­
sorbents and sensors) (Liu et al., 2013; Wang et al., 2013; Zhou et al.,
2010), silica nanoparticles (Zhu et al., 2019), polymers (Hu & Mi, 2013;
Murphy et al., 2010), titanium (Steeves et al., 2016), and many other
matrices. Moreover, melanin-like coatings enable the secondary
coupling reactions with different organic molecules, due to the presence
of catechol domains in their structure which can react with thiols and
amines via Michael addition or Schiff base reactions (Burzio & Waite,

2000; LaVoie et al., 2005). Therefore, the number of scientific reports

concerning this topic grows rapidly, indicating the enormous interest in
this useful technique.
Most scientific reports state that melanin-like coatings are formed
from dopamine. Very rarely levodopa is used for this purpose instead of
dopamine, forming poly(L-DOPA). Both dopamine and levodopa belong
to catecholamine family. Dopamine (3-hydroxytyramine; 2-(3,4-dihy­
droxyphenyl)ethylamine; 4-(2-Aminoethyl)-1,2-benzenediol) contains
both catechol and amino groups in its molecule. Levodopa (L-DOPA;
DOPA; 3,4-Dihydroxy-L-phenylalanine; L-3-Hydroxytyrosine) is closely
related to dopamine (as its precursor in catecholamine synthesis
pathway) and structurally differs from this compound by the presence of
carboxyl group in aminoethyl moiety. Both dopamine and L-DOPA
polymerize in the presence of oxidants (as O2 or Cu2+ ions) and in
slightly alkaline buffers (e.g. in 10–50 mM Tris pH 8.5), although
Bernsmann et al. (Bernsmann et al., 2011) reported that O2 as oxidant
may be not effective in case of L-DOPA. In fact, although dopamine is
used much more frequently as a monomer for polycatecholamine coat­
ings formation, both these compounds are commonly called “dopamine”
in numerous scientific reports. In comparison with PDA, little is known
about poly(levodopa) (poly(L-DOPA)) properties, both in relation to its
biological safety and potential in coupling with attractive biological

* Corresponding author.
E-mail address: (A. Belcarz).
/>Received 8 June 2021; Received in revised form 12 July 2021; Accepted 22 July 2021
Available online 24 July 2021
0144-8617/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( />

A. Michalicha et al.

Carbohydrate Polymers 272 (2021) 118485

molecules. However, carboxyl group in L-DOPA, which is absent in
dopamine molecule, may affect its polymerization to poly(L-DOPA) and
also the polymer properties. For example, presence of carboxyl group (of
approx. 2–3 pKa value) is responsible for negative charge of L-DOPA
which increases the poly(L-DOPA) dispersity in water (Hashemi-Mog­
haddam et al., 2018). Polyolefin membranes coated with poly(L-DOPA)
showed notable content of free –COOH groups on their surfaces and also
an increased hydrophilicity, although the latter was higher for
polydopamine-coated membranes (Xi et al., 2009). Poly(L-DOPA) films
deposited on polypropylene, nylon and poly(vinylidenefluoride) sub­
strates showed significantly higher stability in strong acidic conditions
than analogous polydopamine coatings (Wei et al., 2013). L-DOPA, due
to –COOH content, was also used for the synthesis of PDE-DOPA4
monomer blocks which were further oxidized (with assistance of NaIO4)
to form quickly setting adhesive hydrogel (Burke et al., 2007).
Importantly, free carboxyl group in poly(L-DOPA) molecule could be
an additional site for interactions between poly(L-DOPA) coatings and
different ions/particles/molecules. As suggested by Bernsmann et al.
(Bernsmann et al., 2011), L-DOPA during its polymerization to poly(LDOPA) is turned to 5,6-dihydroxyindole-2-carboxylic acid – an inter­
mediate which is unable to undergo a 2,2′ -branching. Thus, this
carboxyl group in 5,6-dihydroxyindole-2-carboxylic acid units is prob­
ably not engaged in the formation of covalent bonds in poly(L-DOPA)
coatings. Poly(L-DOPA) was already effectively used to attach paclitaxel
to core-shell Fe3O4@poly(DOPA) nanoparticles (Hashemi-Moghaddam
et al., 2018) although the exact role of free carboxyl groups in these
nanoparticles was not explained. In nitrogen-doped graphene quantum

dots obtained with assistance of L-DOPA, free surface carboxyl groups
were prone to coordinate with Fe3+ ions, thus facilitating the electron
transport between ions and dots and in consequence enabling the for­
mation of Fe3O4-dots hybrids (Shi et al., 2016). Thus, poly(L-DOPA)
coatings may exert other properties and allow different applications
than PDA coatings.
Recently, our group synthesized PDA-modified high-set curdlan
hydrogels using thermal polymerization method. Curdlan (β-1,3-glucan)
is a polysaccharide of specific gelling properties, high water sorption
capacity, significant elasticity and relatively high mechanical resistance
(Chen & Wang, 2020). It exhibits therefore high potential for design of
wound dressing materials. However, due to the presence of exclusively
hydroxyl groups in repeating glucose units of curdlan backbone, it is
biologically inert and relatively insusceptible to modifications
improving its biological properties and capability to bind therapeuti­
cally active molecules (Cai & Zhang, 2017). PDA coating could be
therefore an excellent method to introduce modifiable domains for
biological improvement of curdlan. We do have demonstrated that PDAmodified hydrogels showed the ability to bind molecules containing free
amino groups (as proved for gentamicin and peroxidase) and exhibited
unchanged mechanical stability, increased soaking capacity, prolonged
antibacterial properties and Fickian-type mechanism of drug release
(Michalicha et al., 2021). These results suggested that PDA-modified
curdlan hydrogel may serve as a carrier of free amino groupscontaining molecules and be used for different purposes, e.g. antibac­
terial hydrogels for wound dressings.
In view of this, we hypothesized that also levodopa may effectively
form the biologically safe deposits on polysaccharide matrix and may be
used for fabrication of wound dressings. Therefore, in this paper we
report the fabrication of poly(levodopa)-modified curdlan hydrogels.
The hydrogels were first characterized for stability as well as biological
safety of the deposits during contact with blood, fibroblast cell line,

fibroblast primary culture and zebrafish eggs and larvae, in relation to
their possible application as wound dressings. Second, the modified
hydrogels were coupled with gentamicin and their antibacterial activity
was evaluated.

2. Materials and methods
2.1. Synthesis of poly(L-DOPA)-modified curdlan hydrogels
Curdlan powder (from Alcaligenes faecalis; cat. No. 281–80,531; DP
6790; average Mw 1100 kDa; specific rotation [A]20/D: +30 to +35),
Cl− content < 0.5%, heavy metals content (including Pb) < 0.002%, was
provided by Wako Chemicals (Japan); Tris (2-Amino-2-(hydroxymethyl)
propane-1,3-diol) and L-DOPA (3,4-Dihydroxy-L-phenylalanine) by
Sigma-Aldrich (USA). Control and poly(L-DOPA)-modified hydrogels
were synthesized according to procedures described elsewhere
(Michalicha et al., 2021), briefly:
2.1.1. With L-DOPA monomer added to curdlan suspension Before thermal
Gelling (BG)
Suspension of 0.4 g curdlan in 5 ml 10 mM Tris/HCl buffer pH 8.5
was combined with 10 mg (2-LD-BG) or 20 mg (4-LD-BG) of L-DOPA,
stirred 10 min until L-DOPA was completely dissolved, transferred into
glass tubes (ø 13 mm) and polymerized at 93 ◦ C for 15 min. After
cooling, hydrogel was cut into 3 mm slices and incubated in air (air
oxygen as an oxidant) for 24 h at 25 ◦ C to allow L-DOPA polymerization.
Then slices were washed 10 times in 100 ml DI H2O, frozen and
lyophilized (SRK, System Technik GMBK, Germany).
2.1.2. With L-DOPA monomer added to curdlan suspension After thermal
Gelling (AG)
Suspension of 0.4 g curdlan in 5 ml 10 mM Tris/HCl buffer pH 8.5
was transferred into glass tubes (ø 13 mm) and polymerized at 93 ◦ C for
15 min. Cooled hydrogel was cut into 3 mm slices, immersed in 5 ml

10 mM Tris/HCl buffer pH 8.5 containing 10 mg (2-LD-AG) of L-DOPA
and incubated in orbital shaker for 24 h at 25 ◦ C with the access to air
(air oxygen as an oxidant), to allow L-DOPA polymerization. Then slices
were washed 10 times in 100 ml DI H2O, frozen and lyophilized.
2.1.3. Control curdlan hydrogels were prepared as in Section 2.1.2 without
the immersion in L-DOPA solution and further incubation
Prior to cell cultures, zebrafish, drug release and antibacterial ac­
tivity experiments, all curdlan hydrogels were sterilized by ethylene
oxide method in paper/plastic peel pouch (sterilization for 1 h at 55 ◦ C,
aeration for 20 h).
2.2. Gentamicin immobilization and quantitative analysis
Immobilization of gentamicin into modified curdlan samples was
performed by incubation of lyophilized slices in 1 mg/ml gentamicin
(Sigma-Aldrich, USA) in Britton-Robinson buffer pH 8.5, in proportion
33.3 ml of antibiotic solution/1 g lyophilized curdlan hydrogel slices,
using DTS-4 shaker (100 rpm), 24 h at 25 ◦ C, followed by 24 h at 4 ◦ C.
Then slices were washed twice in 50 ml DI water, frozen and lyophilized.
In case of pilot experiment, EDC/NHS activation of poly(L-DOPA)
carboxyl groups was used for binding with gentamicin. Hydrogel sliced
were first soaked in 0,1 M MES buffer pH 6,5 and then incubated in
mixture of 0.1 M EDC (N-(3-Dimethylaminopropyl)-N-ethyl­
carbodiimide hydrochloride; Sigma-Aldrich, USA) and 0.2 M NHS (Nhydroxy succinimide; Sigma-Aldrich, USA) in 0,1 M MES buffer pH 6,5
for 1 h, at 25 ◦ C, on plate shaker DTS-4 (ELMI, USA), 100 rpm. After­
wards, the samples were washed twice (10 min.) in distilled water and
immersed in 1 mg/ml gentamicin (Sigma-Aldrich, USA) in 0,05 M
NaHCO3 (pH 8.5), in proportion 33.3 ml of antibiotic solution/1 g
lyophilized curdlan hydrogel slices, using DTS-4 shaker (100 rpm), 24 h
at 25 ◦ C.
Gentamicin concentration in solutions before and after incubation
was evaluated according to Ginalska et al. (Ginalska et al., 2004), based

on gentamicin derivatization by phthaldialdehyde (Sigma-Aldrich,
USA). Amount of immobilized gentamicin was calculated from formula
(1):
2


A. Michalicha et al.

Ci(µg/g d.w.)

[
]
Cb(µg/ml) –Ca(µg/ml) x V(ml)
=
Mg

Carbohydrate Polymers 272 (2021) 118485

appropriate calibration curve (using 96-well plates and Synergy H4
hybrid microplate reader, Biotek, USA) and were 136 mg% and
0.18 mg/ml, respectively. For hemolysis test, lyophilized hydrogel slices
(30 mg ± 2 mg) were immersed in 2 ml of blood 100× diluted in PBS
pH 7.4 without Ca2+ and Mg2+ ions. Positive control contained 0.1%
Triton X-100 while negative one: 30 mg ± 2 mg of HDPE (high density
polyethylene, Sigma-Aldrich, USA). Then samples were incubated 3 h at
37 ◦ C in Innova 42 incubator shaker (New Brunswick Scientific, USA),
150 rpm. Erythrocytes-released hemoglobin was estimated using reac­
tion with Drabkin reagent, as above. For blood clot formation test, 100 μl
of whole 10 mM CaCl2-activated blood was placed onto 30 mg ± 2 mg
lyophilized hydrogel slices or pieces of HDPE (negative control). Nonactivated Ca2+-free whole blood (100 μl) served as positive control.

Then samples were incubated 15 min., 30 min. or 45 min. at 37 ◦ C,
without shaking (controls were performed individually per each time
point). Then all samples were incubated with 2.5 ml of distilled water for
5 min. Finally the hemoglobin content in solution was estimated using
reaction with Drabkin reagent, as described above. Each experiment was
performed in triplicate. Statistically significant differences between
negative control and various samples were considered at p < 0.0001,
according to a One-way ANOVA with post-hoc Dunnett’s test (GraphPad
Prism 8.0.0 Software, San Diego, CA).

(1)

where:
Ci(μg/g d.w.) – amount of gentamicin immobilized on samples (μg/g of
dry hydrogel weight).
Cb(μg/ml) – concentration of gentamicin in solution before incubation
with samples (μg/ml).
Ca(μg/ml) – concentration of gentamicin in solution after incubation
with samples (μg/ml).
V(ml) – volume of solution incubated with samples (ml).
Mg – dry weight of samples (g).
2.3. Characterization of modified curdlan hydrogels
FTIR-ATR spectra were collected using Vertex 70 IRspectrophotometer (4000–400 cm− 1, 64 scans, 4 cm− 1 spectral resolu­
tion; Bruker, USA) and analysed using OPUS 7.0 software (Bruker, USA).
X-ray photoelectron spectroscopy (XPS) measurements were made
on a Microlab 350 (Thermo Electron) spectrometer with nonmonochromatic AlKα (hν = 1486.6 eV, power 300 W, voltage 15 kV)
radiation as an X-ray excitation source. A lateral resolution was about
0.2 cm2. The high-resolution XPS spectra were acquired using the
following parameters: pass energy 40 eV, energy step size 0.1 eV. A
Smart function of background subtraction was used to obtain the XPS

signal intensity. The all collected XPS peaks were fitted using an
asymmetric Gaussian/Lorentzian mixed function. The measured binding
energies were calibrated with respect to the energy of C 1s at 285.0 eV.
Avantage-based data system software (Version 5.9911, Thermo Fisher
Scientific) was used for data processing.
For evaluation of soaking capacity, lyophilized curdlan hydrogels
were incubated in 5 ml 0.9% NaCl at 37 ◦ C for up to 72 h. In defined time
points, the samples were withdrawn from the liquid, drained on What­
man paper to remove the excess of the liquid and weighed (XS205,
Mettler-Toledo, Switzerland). The obtained data were normalized (to
100% of initial dry weight of samples). Experiment was performed in
triplicate. Statistically significant differences between non-activated and
activated sample in each modification mode were considered at
p < 0.05, according to Student’s t-test (GraphPad Prism 6 Software, San
Diego, CA).

2.6. Cell culture experiments
Cell culture tests were conducted using human skin fibroblasts:
normal human skin fibroblast cell line (BJ cell line, ATCC-LGC Stan­
dards) and primary human dermal fibroblasts (HDFs, ATCC-LGC Stan­
dards). Fibroblasts were cultured at 37 ◦ C in a humidified atmosphere of
5% CO2 and 95% air. BJ fibroblasts were maintained in EMEM medium
supplemented with 10% fetal bovine serum (FBS, Pan-Biotech), and
100 U/ml/100 μg/ml penicillin/streptomycin mixture (Sigma-Aldrich
Chemicals). HDFs were maintained in a Fibroblast Basal Medium sup­
plemented with the components of Fibroblast Growth Kit-Low Serum
(both purchased from ATCC-LGC Standards).
2.6.1. Cytotoxicity tests according to ISO 10993-5 standard
Fibroblast suspension with a concentration of 2 × 105 cells/ml was
seeded in 100 μl into the wells of 96-multiwell plates. After 24-h incu­

bation, the culture medium was discarded and the monolayer of cells
was exposed to the extracts of the tested samples. The extracts of the
materials were prepared according to ISO 10993-12 standard by placing
0.1 g sample in 1 ml of complete culture medium followed by 24-h in­
cubation at 37 ◦ C. Non-toxic extract serving as a negative control of
cytotoxicity was prepared by the incubation of complete culture me­
dium in a polystyrene vessel for 24 h at 37 ◦ C without any biomaterial
(extract marked as PS control). Fibroblasts were maintained in the ex­
tracts for 48 h and then cytotoxicity of the samples was determined by
evaluation of cell metabolism using MTT assay (Sigma-Aldrich Chem­
icals) and cell number using total LDH test (Sigma-Aldrich Chemicals).
The MTT assay was carried out based on the procedure described earlier
(Przekora et al., 2014). Total LDH test was performed after cell lysis
according to the manufacturer instructions. Results of MTT and total
LDH tests were presented as the percentage of negative control of
cytotoxicity (100% viability in terms of cell metabolism and cell num­
ber). Three independent experiments were conducted for both cyto­
toxicity tests. Statistically significant differences between negative
control (PS control) and various samples were considered at p < 0.05,
according to a One-way ANOVA with post-hoc Dunnett’s test (GraphPad
Prism 8.0.0 Software, San Diego, CA).

2.4. Stability of poly(L-DOPA) deposits
Poly(L-DOPA)-modified hydrogel slices were extensively washed in
distilled water (10 times in 0.5 l; first 5 washes for 2 h, second 5 washes
for 12 h; RM 5-30 V shaker (CAT M. Zipperer Gmbh, Germany),
30 rpm.). Incubation in human serum (kindly donated by Regional
Center of Blood Donation and Blood Treatment in Lublin) and in human
blood (collected after approval of Bioethics Committee at the Medical
University of Lublin, no KE-0254/258/2020) was performed (2 ml per

30 mg of dry hydrogel weight) on plate shaker DTS-4 (ELMI, USA),
100 rpm, at 37 ◦ C, for up to 96 h; then washed once in distilled water.
Effect of pH was tested using 0.1 M Britton-Robinson buffers pH 2, 4, 6,
8, 10 and 12 (the same liquid-to-biomaterial proportion as for human
serum and blood), for at 37 ◦ C, for 7 days. Macro photography of
plasma-, blood- and buffers-incubated hydrogels and post-incubation
buffers was performed using E-520 digital camera (Olympus, Japan).
2.5. Hemolysis and blood clot formation test

2.6.2. Cell proliferation
Fibroblast suspension with a concentration of 1.5 × 104 cells/ml was
seeded in 100 μl into the wells of 96-multiwell plates. After 24-hour
incubation, total LDH test was conducted to determine cell number at
starting point (time = 0 h – before addition of the extracts). The exact

Human blood was collected on citrate from healthy volunteer on
approval of Bioethics Committee at the Medical University of Lublin, no
KE-0254/258/2020. Its total hemoglobin and plasma hemoglobin con­
centration were estimated on basis of reaction with Drabkin reagent and
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Carbohydrate Polymers 272 (2021) 118485

number of fibroblasts was calculated using calibration curve prepared
for known concentrations of BJ cells and HDFs. Then, extracts of the
biomaterials (prepared as described in section 2.6.1) were added to the
cells which were further incubated for 3 days. Cell number was deter­

mined after 24- and 72-hour exposure to the extracts. Three independent
proliferation tests were performed. Statistically significant diffrences
between PS control (non-toxic control extract prepared by incubation of
culture medium in a polystyrene vessel) and various samples were
considered at p < 0.05, according to a One-way ANOVA with post-hoc
Dunnett’s test (GraphPad Prism 8.0.0 Software; San Diego, CA).

ZEISS, Germany). Incidence of morphological and physiological abnor­
malities e.g. a lack of somite formation, scoliosis, or the pericardial
oedema were observed and compared to the control embryos. Image
analysis was performed to determine the percentage of malformed
embryos.
2.7.3. Locomotor activity assay
Test was performed on 5 day post fertilization (dpf) larvae. One larva
per well was placed in 96 multi-well plate. To each well, either 200 μl
extract of control or poly(L-DOPA)-modified hydrogels or the same
volume of pure E3 medium was added (each group of n = 20) and the
zebrafish larvae were incubated in the extracts for 30 min before the
test. Then, EthoVision XT 15 video tracking software (Noldus Informa­
tion Technology b.v., The Netherlands) was used for evaluation of lo­
comotor activity. The distance moved in 10 min period was calculated in
cm, in a light condition. The results were processed by the one-way
ANOVA analysis with Dunnett’s post hoc test. A p value <0.05 was
considered statistically significant. All statistical analyses were per­
formed using GraphPad Prism 8 (GraphPad Software, San Diego, CA,
USA).

2.6.3. Cell adhesion to the samples
Before the experiment, all samples in the form of discs (12 mm in
diameter and 2 mm in height, weighing 30 mg ± 2 mg) were sterilized

by ethylene oxide, placed in the wells of 24-multiwell plate and soaked
in the complete culture medium. Then, fibroblasts (2 × 105 cells per
sample) were seeded directly on the top surface of the materials. Fi­
broblasts seeded into a polystyrene well of the 24-multiwell plate served
as control cells (PS control), revealing good adhesion and high viability.
After 72-h culture of fibroblasts on the samples, their adhesion and
viability were evaluated using Live/Dead Double Staining Kit (SigmaAldrich Chemicals) and confocal laser scanning microscope (CLSM,
Olympus Fluoview equipped with FV1000). Cell staining was carried out
according to the manufacturer protocol, using two fluorescent probes:
calcein-AM (green fluorescence of viable cells) and propidium iodide
(red fluorescence of dead cells).

2.8. Gentamicin release
Drug release evaluation was performed as described earlier
(Michalicha et al., 2021), both in semi-open-loop and close-loop sys­
tems. Both procedures were described below:

2.7. Danio rerio embryotoxicity tests

2.8.1. Semi-open-loop system
Samples (in triplicate) were incubated with sterile PBS pH 7.4
(proportion: 1 ml PBS/0.1 g dry weight of hydrogel sample) at 37 ◦ C,
with agitation (50 rpm, Innova 42, New Brunswick Scientific, USA). The
extracts were collected daily and replaced by the same volume of PBS.
Gentamicin concentration was measured in daily collected extracts,
until detectable.

2.7.1. Zebrafish maintenance
Danio rerio of the AB strain (Experimental Medicine Centre, Medical
University of Lublin, Poland) were maintained at 28.5 ◦ C, on a 14/10 h

light/dark cycle, under standard aquaculture conditions. Fertilized eggs
were collected via natural spawning. Embryos were reared in E3 embryo
medium (pH 7.1–7.3; 17.4 μM NaCl, 0.21 μM KCl, 0.12 μM MgSO4 and
0.18 μM Ca(NO3)2) in an incubator (IN 110 Memmert GmbH, Germany)
at 28.5 ◦ C.
For the experiments, the embryos were treated with hydrogel ex­
tracts prepared by incubation of the control and poly(L-DOPA)-modified
hydrogels in sterile E3 medium (in proportion: 1 ml E3 medium/0.1 g
dry weight of hydrogel sample) at 37 ◦ C, 24 h, with agitation (50 rpm,
Innova 42, New Brunswick Scientific, USA). The extracts were therefore
prepared in analogous manner as in tests of cytotoxicity (Section 2.6.2.)
as well as drug release and antibacterial activity in semi-open-loop
system (Sections 2.8.1. and 2.9.3), to enable the appropriate compari­
son between therapeutic potential and biological safety of hydrogels.
Immediately after the experiment, larvae were killed by immersion
in 15 μM tricaine solution. All experiments were conducted in accor­
dance with the National Institute of Health Guidelines for the Care and
Use of Laboratory Animals and the European Community Council
Directive for the Care and Use of Laboratory Animals of 22 September
2010 (2010/63/EU).

2.8.2. Closed-loop system
USP 4 Flow-through cell dissolution testing was performed using CE1
Sotax units (Donau Lab, Switzerland) for gentamicin-loaded control and
poly(L-DOPA)-modified hydrogels, each in amount containing 2 mg of
gentamicin, with 50 ml PBS pH 7.4 and 1 ml/min laminar flow rate, at
37 ◦ C. 3 ml of extracts were collected at defined time intervals for the
estimation of drug concentration (3 ml of fresh PBS was immediately
added to the units to maintain the constant volume). Cumulative drug
concentrations were calculated based on the results of 4 independent

experiments (each in triplicate). Korsmeyer–Peppas and Higuchi models
were used to describe the drug release kinetics. Korsmeyer–Peppas
model was also used to determine the drug release mechanisms, ac­
cording to a general equation:
Mt /M∞ = ktn
where:
Mt is the amount of drug released from the composite in time t, M∞ is
the accumulated released drug amount at time t → ∞, k and n are the
kinetic constant and the release exponent, respectively.
Drug release mechanism was interpreted via nonlinear regression
analysis, using the Statistica 10 software.

2.7.2. Fish embryo toxicity (FET) test
The collected embryos were transferred to a Petri dish with E3 me­
dium and then placed in 96-well plates, 1 embryo per well. To each well,
either 200 μl extract of control or poly(L-DOPA)-modified hydrogels or
the same volume of pure E3 medium was added (each group of n = 15).
The embryos were then maintained in the incubator (as described in
Section 2.7.1). Apical observations of acute toxicity in zebrafish em­
bryos 24–96 h post fertilization (hpf) were performed according to
OECD guidelines for the testing of chemicals no 236 (Organization for
Economic Co-operation and Development. 2013. Test No. 236: Fish
embryo acute toxicity (FET) test. Guidelines for the Testing of Chem­
icals. Paris, France). Coagulation, somite formation, tail detachment and
heartbeat were observed using a stereomicroscope (Zeiss Axio Vert,

2.9. Antibacterial activity evaluation
Antibacterial activity evaluation was performed as described earlier
(Michalicha et al., 2021), briefly:
2.9.1. Bacterial strains

3 reference bacterial strains (Staphylococcus aureus ATCC 25923,
Staphylococcus epidermidis ATCC 12228 and Escherichia coli ATCC
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Carbohydrate Polymers 272 (2021) 118485

25922) were refreshed from sterile microbanks using Mueller-Hinton
Agar medium (Oxoid, USA), at 37 ◦ C for 24 h. Then bacteria were
transferred into Mueller-Hinton (M-H) broth medium (Oxoid, USA) and
cultured for 24 h at 37 ◦ C. Directly before the experiments, the sus­
pension of propagated bacteria was diluted to appropriate density. The
strains were chosen on basis of available statistics of infections occur­
rence for implantable biomaterials (Khatoon et al., 2018).

NaCl (50 ml, 4 times). Washed samples were incubated with Viability/
Cytotoxicity Assay Kit for Bacteria Live & Dead Cells (Biotium, USA) in
0.9% NaCl (according to manufacturer instructions: at R/T, 15 min, in
darkness). After staining, the samples were washed in 0.9% NaCl to
remove non-absorbed dye. Adhered bacteria were visualized by confocal
microscopy (Olympus Fluoview FV1000; Olympus, Japan).
3. Results and discussion

2.9.2. Antibacterial activity test (based on the standard: AATCC Test
Method 100–2004)
Briefly, gentamicin-loaded hydrogels, both control and poly(LDOPA)-modified ones, were placed on sterile Petri dishes (in quadru­
plicate) and treated with bacterial suspension (1.5 × 105 CFU/ml) of
each strain, prepared in M-H broth (250-fold in sterile 0.9% NaCl). The

volumes of bacterial suspension were calculated to be completely
absorbed by hydrogels without the excess leaching outside. Hydrogel
samples were then incubated at 37 ◦ C for 24 h (T24h), then transferred to
sterile 0.9% NaCl (in volume 100-fold larger than volume of bacterial
suspension absorbed by samples) and vigorously shaken (1 min.) to elute
the bacterial cells. Samples of the collected eluate were plated (in trip­
licate) onto M-H agar using automatic plater (EasySpiral Dilute, Inter­
science, France). Another set of untreated control hydrogels was soaked
in bacterial inoculate as above and immediately (T0h) subjected to
bacterial cells elution. M-H agar plates with plated bacteria eluted from
hydrogel samples were incubated at 37 ◦ C for 48 h. CFU were then
counted for each plate and percent reduction of bacteria was calculated
by the formula (2):
%reduction = [(B − A)B ]*100

L-DOPA monomer was used for modification on curdlan fibers ac­
cording to two different modes, as presented in procedure description:
the monomer was introduced to curdlan before (BG; Before Gelling) or
after (AG; After Gelling) its thermal gelling into high-set hydrogel
(Fig. 1a). Hypothetically, when monomer solution is added to already
polymerized curdlan hydrogel (AG method), poly(L-DOPA) can be
formed on the surface of triple helix curdlan network (Zhang & Edgar,
2014). Whereas when L-DOPA monomer is added to curdlan suspension
before its thermal gelling (BG method), it may enter the fibers of nonpolymerized curdlan and then polymerize not onto but within the
further formed triple helix structure of curdlan hydrogel. This difference
may affect the structure and therefore properties of poly(L-DOPA) de­
posits on hydrogel helical fibers. The concentration of L-DOPA monomer
used in this study for poly(L-DOPA) modification was 2 or 4 mg/ml. Most
commonly, concentration of catecholamine monomer used for poly­
merization in limited to 2 mg/ml solution, because higher monomer

concentrations may result in the formation of unstable coatings (Liu
et al., 2018). However, conditions of L-DOPA polymerization according
to BG method are different than standard catecholamine polymerization
(by incubation of solid samples in monomer solution). Therefore, in this
particular method, two L-DOPA concentrations were tested (2 mg/ml
and 4 mg/ml). Uniform brownish-black color of all synthesized poly(LDOPA)-modified curdlan hydrogels (2-LD-BG, 4-LD-BG and 2-LD-AG)
indicated their efficient covering by polycatecholamine layer (Fig. 2a).
One of important features of hydrogels is their ability to absorb liq­
uids (soaking capacity, water absorption capacity). It was found earlier
that wettability of biomaterials can be improved by coating with PDA
(Guo et al., 2016; Li et al., 2020; Liu et al., 2018). Therefore, this
property was evaluated for poly(L-DOPA)-modified curdlan hydrogels.
All versions of modified curdlan hydrogels reached the total soaking
capacity after 48 h. Interestingly, only the value of total soaking capacity
for 2-LD-AG material was increased by 13% in comparison with other
samples (811% for 2-LD-AG and 707–711% for 2-LD-BG, 4-LD-BG and
control curdlan hydrogels), referred to their initial dry weight (Fig. 1c).
Therefore, the increased soaking capacity of poly(L-DOPA)-modified
curdlan seems to be related to the specific mode (After Gelling) of LDOPA monomer introduction into curdlan matrix.
Chemical structure of the samples was tested by FTIR technique, in
reference to pure L-DOPA monomer. Pure poly(L-DOPA) was impossible
to be obtained in relevant polymerization conditions, neither in solution
nor on glass slides. This phenomenon, due to the high dispersity of poly
(L-DOPA), was in agreement with previous reports (Bernsmann et al.,
2011; Hashemi-Moghaddam et al., 2018). The results of FTIR analysis of
produced hydrogels were similar to those earlier obtained for PDAmodified curdlan hydrogels (Michalicha et al., 2021). First, no signs of
− 1
L-DOPA, manifested by the presence of N–H stretching (3393 cm ),
− 1
–

–
N H bending in primary amine (1526 cm ), C N stretching
(1282 cm− 1) and C–H out-of-plane stretching (816 cm− 1) bands,
respectively (Mohanraj et al., 2013; Zhou et al., 2013), were detected in
modified hydrogels (Fig. 1b). This can be caused by low quantity of poly
(L-DOPA) deposited within the curdlan hydrogel: 2 or 4 mg of L-DOPA
monomer added to 400 mg of curdlan corresponds to less or equal to 1%
of total dry hydrogel weight. Therefore this amount of poly(L-DOPA)
distributed within the curdlan hydrogel matrix was probably too low to
be detected by FTIR technique. Second, spectra of 2-LD-BG, 4-LD-BG and
2-LD-AG were similar to that of control curdlan. This suggests the lack of
chemical changes in basal curdlan hydrogel structure.

(2)

where:
B – the number of bacteria recovered from the inoculated control
specimen immediately after the inoculation (T0h);
A – the number of bacteria recovered from the inoculated poly(LDOPA)-modified specimen 24 h after the inoculation (T24h).
2.9.3. Bacterial growth inhibition in semi-open-loop system
Lyophilized and sterilized control and poly(L-DOPA)-modified
hydrogels were incubated in sterile M-H broth (proportion: 1 ml me­
dium/0.1 g dry weight of hydrogel sample) at 37 ◦ C, with agitation
(50 rpm, Innova 42, New Brunswick Scientific, USA). The extract was
collected daily and replaced by the same volume of fresh medium. The
experiment was performed in triplicate, until the loss of antibacterial
properties in collected extract was observed.
200 μl of each collected extract (in quadruplicate) in 96-well plate
was inoculated by 10 μl of inoculate of three bacterial strains (density:
3 × 107 CFU (colony forming units)/1 ml) and cultured for 24 h at 37 ◦ C,

on plate shaker DTS-4 (ELMI, USA), 100 rpm. In parallel, bacterial
growth positive control was tested for each strain in M-H broth, inocu­
lated and treated as above. After 24 h, absorbance (at 660 nm) of the
cultures was measured on Synergy H4 Hybrid microplate reader (Bio­
Tek, USA). Inhibition of bacterial growth by hydrogel extracts was
calculated as a percent of growth in positive controls. Beginning of
bacterial infection was noted when the density in inoculated extracts
was higher than that of untreated blank (pure M-H broth). Experiment
was performed until bacterial growth in inoculated extracts reached the
optical density of positive growth controls (final stage of infection).
Hydrogel samples were finally subjected to the test of bacterial adhesion
(as described in Section 2.9.4.).
2.9.4. Bacterial adhesion
The samples of hydrogel after the bacterial growth inhibition test
were incubated in 1 ml of bacterial suspensions (approx. 1.0 × 108 cells/
ml; in M-H broth) of each strain, for 1.5 h, at 37 ◦ C, without shaking.
Afterwards, non-adhered bacteria were gently washed away with 0.9%
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Carbohydrate Polymers 272 (2021) 118485

Fig. 1. Description (a), FTIR spectra (b) and soaking capacity (c) of synthesized curdlan hydrogel samples. LDOPA – L-DOPA monomer. Soaking capacity was
expressed as % of hydrogels weight increase.

Deeper insight into chemical structure of modified hydrogels sam­
ples was provided by XPS technique which was used to determine the
elemental composition and chemical state information of the surface

elements present in the investigated samples. The control and modified
samples consisted mainly of C, O and N with smaller contribution of
elements such as Ca and Si. Table 1 presents the calculated chemical
composition of the investigated samples. The atomic ratio of C to O at
the surface of control sample was around 1.8 and differs significantly for
the values obtained for 2-LD-BG (C/O = 5.6) and 2-LD-AG (C/O = 2.2),
while remained similar for 4-LD-BG (C/O = 1.7). The nitrogen contri­
bution in control sample is approximately 0.3 at.%. For the modified
samples the nitrogen was detected only for 2-LD-BG (0.1%) and 2-LD-AG
(1.3%).
High-resolution spectra of the C1s, O1s and N1s regions obtained for
control and modified samples are presented as Supporting Information.
The binding energies and assigned functional groups for each peak
within the region envelope are shown in Fig. S1. The percent contri­
bution for each functional group within the C1s, O1s and N1s regions for
control and modified samples are presented in Table 1. In general, the
peak energies of each component remained similar but the relative in­
tensities of each component varied, particularly for 4-LD-BG and 2-LDAG samples.
The C1s region was fit with four peaks assigned to C–C/C–H, C–O/
– O and O–
– C–OH species with the dominant peak being
C–N, C–
attributed to C–O and C–C species. The contribution of C–O species
varied from 59.3% to 64.4% with the highest value obtained for control
sample. The C–C/C–H feature increased in relative intensity upon
modification (13.5–24.9%) comparing to control sample (13.2%). The
– O and
significant difference in contribution one can see for C–
– C–OH components. The C–
– O species were not detected for 2-LDO–

– C–OH group were not present for 4-LD-BG (Table 1).
AG, while the O–
– C, O–C and
The O1s region was fit with three peaks assigned to O–
adsorbed water; except for 4-LD-BG for which water was not detected.
The contribution of each species differs depending on the modification
conditions. The dominant peak was attributed to O–C species or all
samples under investigation. It reached the highest contribution for the
control sample (93.7%) and decreased for modified samples
(82.7–87.1%) (Table 1).
The N1s region for control and 2-LD-BG samples presented one
component which can be assigned to secondary amine (R–NH–R). The
N 1s spectrum of 2-LD-AG showed three contributions arising from
primary (R-NH2), secondary (R–NH–R) and tertiary/aromatic
– N–R) amine functionalities. According to the literature and probable
(–
reaction mechanism of poly(L-DOPA) formation (Azari et al., 2014;
Manolakis & Azhar, 2020; Zhao et al., 2019), the primary amine is
associated with L-DOPA and the secondary and tertiary amine is asso­
ciated with both polymerization reaction intermediates and poly(LDOPA).
Presence of nitrogen (0.3 at.%) and carboxyl groups (2.8% of C1s) in
control hydrogel can be easily explained by 1.6% content of protein in
´dkov´
´ et al.,
curdlan which was confirmed by Hroma
a (Hrom´
adkova
2003). Increase of nitrogen (1.3 at.%) and carboxyl groups (13% of C1s)
content in 2-LD-AG sample results from the deposition of poly(L-DOPA)

on the surface of triple helical curdlan network. However, the expla­
nation of results for samples obtained according to BG method is more
difficult, especially for 4-LD-BG. For this particular sample, neither ni­
trogen nor carboxyl groups were detected by XPS although stable black
color of the sample confirmed the presence of poly(L-DOPA) in hydrogel.
There are two possible explanations of this phenomenon, due to the
specific method of samples synthesis (in which L-DOPA monomer is
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Carbohydrate Polymers 272 (2021) 118485

Fig. 2. Stability of control and poly(L-DOPA)-modified curdlan hydrogel samples: before (BW) and after (AW) extensive washing in water (a); after incubation (24 h
and 96 h) in human serum or blood (b); after incubation (7 days) in Britton-Robinson buffers pH 2–12 (c).
Table 1
Chemical composition and XPS functional group percentages for control and modified curdlan hydrogels.
Chemical composition in at. %
Sample

at.%

Control
2-LD-BG
4-LD-BG
2-LD-AG

C/O

C

O

N

Ca

Si

64.3
84.7
62.8
67.7

35.0
15.2
37.2
30.9

0.3
0.1
–
1.3

0.1
–
–
0.1

0.4
–
–
–

1.8
5.6
1.7
2.2

XPS functional group percentages
Sample
Control
2-LD-BG
4-LD-BG
2-LD-AG

C1s

O1s

N1s

C–C, C–H

C–O, C–N

–O
C–

–C–OH
O–

–C
O–

O–C

H2O

–
–N–R

R–NH–R

R–NH2

13.2
13.5
20.2
24.9

64.4
59.3
63.2
62.1

17.6
22.6
10.4

–

2.8
4.6
–
13.0

2.9
2.5
14.3
6.5

93.7
82.7
85.7
87.1

3.4
14.8
–
6.3

–
–
–
5.6

100.0
100.0
–

88.0

–
–
–
6.4

added to hydrated curdlan suspension before its thermal gelation at
93 ◦ C; Section 2.1.1.). One of them is partial thermal decomposition of LDOPA resulting in a loss of carboxyl group (in particular in 4-LD-BG
sample). However, thermal studies on L-DOPA showed that this com­
pound is stable in the temperatures lower than 250 ◦ C (Ledeti et al.,
2016). Second explanation is more probable and is a consequence of
hydrogel synthesis method. Electron microscopy of aqueous curdlan
suspension revealed the presence of fibrillary structures, with

microfibrils of 10–20 nm in width (when heating temperature was
90 ◦ C) or of 30–40 nm in width (when heating temperature was 120 ◦ C)
(Kasai & Harada, 1980). If L-DOPA was entrapped within these micro­
fibrils and poly(L-DOPA) was formed afterwards (as in BG method), it
could be undetectable for XPS technique (penetration depth of the Xrays is only few nanometers). In AG method, poly(L-DOPA) is more likely
to be deposited on the surface of curdlan microfibrils than inside them.
Thus, it is possible that steric location of poly(L-DOPA) within curdlan
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Carbohydrate Polymers 272 (2021) 118485

hydrogels obtained by BG and AG methods is different and this may

affect their properties.
Stability of poly(L-DOPA) deposits on curdlan hydrogels was studied
in different conditions. First, we observed that the modification was
stable after very extensive wash in water (5 l of water in toto, 2 h or 12 h
long washes) (Fig. 2a). Second, effect of human serum and fresh human
blood was verified, because wound dressings are likely to contact not
only with skin but also with serum or whole blood. Both blood and
serum contain a variety of enzymes which may decompose poly(LDOPA) polymer. However, 24 h and even 96 h incubation of poly(LDOPA)-modified hydrogels with these liquids did not cause any visible
change of polycatecholamine deposits, as indicated by stable black color
of the samples (Fig. 2b). Serum and blood themselves did not visibly
change neither (data not shown). Thus, enzymatic pattern of these body
liquids seems not to affect the stability of poly(L-DOPA) modification.
Third, impact of pH on poly(L-DOPA)-modification was tested in buffers
of pH ranging from 2 to 12. After 7 days of incubation, the modified
hydrogels seemed to be stable in each pH; however, the buffers them­
selves became slightly brownish in pH 10 and 12 (Fig. 2c). This sug­
gested that applied modification was slightly unstable in pH 10–12. The
stability of poly(L-DOPA) deposits in acidic and physiological pH (2–8)
which include those typical for healthy and inflamed tissue, suggests
their possible applications in design of wound dressing biomaterials.
Slightly higher lability of studied deposits in alkaline pH range, which
does not appear in physiological conditions in human body, does not
seem to disqualify poly(L-DOPA)-modified curdlan hydrogels for this
purpose.
Contact between wound dressing biomaterials and human blood is
likely in wound treatment. Thus, it is important to define the impact of
such biomaterials on blood compatibility. PDA was earlier reported to
improve the blood compatibility of reduced graphene oxide nano­
particles in comparison with bare graphene oxide (Cheng et al., 2013). It
was therefore interesting to verify the blood compatibility of poly(LDOPA)-modified curdlan hydrogels. First phenomenon which should be

studied is hemolysis which can be monitored by the release of free he­
moglobin (HB) from erythrocytes destroyed upon a contact with tested
materials. As shown in Fig. 3a, the level of HB released from the blood
incubated with poly(L-DOPA)-modified curdlan hydrogels is minimally
(but statistically significantly different) higher than negative control
test. Second phenomenon concerning biomaterials-blood interaction is
clot formation which is an important factor in the process of wound
closure. Clot formation may be monitored by the amount of HB released
from erythrocytes which were not entrapped within the clot network. It
was observed that contact of blood with pristine (control) curdlan
caused a delay in clot formation: the amount of free HB for this sample
was 2–3 times higher than for normal clot (negative). Also, the clotting
process was not complete even after 45 min. However, for all poly(L-

DOPA)-modified curdlan hydrogels the clot formation was at a higher
level as for normal clot (negative). The obtained results are in agreement
with reports that PDA coatings demonstrate good affinity to protein
adsorption and thus may cause blood coagulation (Xie et al., 2017). In
this particular case, poly(L-DOPA) deposits on curdlan hydrogels
normalized the blood clot formation which was negatively affected by
pristine curdlan and increased the blood compatibility of this hydrogel.
Wound dressings designed in this way may help to reduce bleeding and
increase the rate of wound healing.
The proposed application of poly(L-DOPA)-modified curdlan con­
cerns the medical purposes, precisely the synthesis of wound dressings.
Therefore, fibroblast cell lines are the first choice cultures to verify the
hydrogel cytotoxicity. Reference cell line and primary culture of fibro­
blasts were selected for this study. Lack of cytotoxicity of tested samples
was expected because previous reports concerning polydopamine coat­
ings suggested that they are not only neutral for living cells and or­

ganisms but may even reduce the in vivo toxicity of biomaterials (Hong
et al., 2011). However, MTT cytotoxicity test, which is an indicator of
cell metabolism, showed that human skin fibroblasts (both BJ cells and
HDFs) exposed to the extracts of the samples had significantly reduced
metabolism compared to the control cells (PS control) (Fig. 4a). BJ cells
had cell metabolism reduced by 50% after exposure to 2-LD-BG, 4-LDBG, and 2-LD-AG and by 60% after treatment with control extract (pure
biomaterial). Interestingly, sample extracts decreased metabolism of
HDFs by approx. 30% and in the case of 2-LD-AG extract by 20%. It
should be noted that according to ISO 10993-5 standard, results ob­
tained with BJ cells indicated cytotoxicity of the samples, whereas re­
sults obtained with HDFs their non-toxicity since cell viability was
higher or equal to 70% (according to ISO, exposure to 100% extracts
should not reduce cell viability by more than 30%).
Surprisingly, total LDH test, which determines cell number/biomass
compared to the healthy control cells, revealed that only 4-LD-BG
extract was cytotoxic to the fibroblasts as both BJ cells and HDFs had
reduced cell number by approx. 50% (Fig. 4a). According to ISO 109935 standard, 2-LD-BG extract was non-toxic because it decreased cell
number to 85% (BJ cells) and to 70% (HDFs). Importantly, fibroblasts
exposed to 2-LD-AG extract revealed cell biomass near 100%, proving its
high safety and non-toxicity. Obtained results suggested that extracts of
the control, 2-LD-BG, and 2-LD-AG samples negatively affected cell
metabolism, but were non-toxic since cell number was decreased by less
than 30% compared to the control cells.
Cell proliferation assay showed significant acceleration of fibroblasts
proliferation only after their exposure to the extract of 2-LD-AG
(Fig. 4b). Extract of 2-LD-BG increased cell number after 24-hour
exposure time, but further incubation in the presence of this extract
resulted in cell number decrease. Importantly, in the case of HDFs a
significant decrease in cell number was observed after 72-exposure to

Fig. 3. Hemolysis (a) and clot formation (b) in blood incubated with control and poly(L-DOPA)-modified curdlan hydrogel samples. (a) & symbol indicate statis­
tically significant results according to One-way ANOVA with post-hoc Dunnett’s test in comparison with negative test (p < 0.005); value of positive test was reduced
10 fold; (b) *, # and ^ symbols indicate statistically significant results according to One-way ANOVA with post-hoc Dunnett’s test for 15 min., 30 min. and 45 min.,
respectively, in comparison with negative tests for each time point (p < 0.0001).
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Carbohydrate Polymers 272 (2021) 118485

Fig. 4. Cell culture experiments performed with the use of sample extracts, normal human skin fibroblastcell line (BJ), and primary human dermal fibroblasts
(HDFs): (a) Cytotoxicity tests: MTT assay (cell metabolic activity) and total LDH assay (cell number/biomass); PS control – negative control of cytotoxicity revealing
100% viability; asterisks indicate statistically significant results according to One-way ANOVA with post-hoc Dunnett’s test (*** p < 0.001); (b) Cell proliferation
assay; PS control – cells exposed to non-toxic extract prepared by incubation of culture medium in a polystyrene vessel; asterisks indicate statistically significant
results according to One-way ANOVA with post-hoc Dunnett’s test (* p = 0.03, ** p = 0.006, *** p < 0.001).

the 2-LD-BG and 4-LD-BG extracts. Surprisingly, although 4-LD-BG
extract showed cytotoxic effect towards BJ cells reducing their meta­
bolism and number by 50% (Fig. 4a), it did not significantly affect
proliferation of these cells. Observed discrepancy between cytotoxicity
and proliferation assays may be explained by growth phase of the fi­
broblasts during mentioned tests. Cytotoxicity test was performed using
100% confluent culture of the cells, so fibroblasts were then in a sta­
tionary phase and thus were prone to dying and detachment from the
well surface. Whereas proliferation assay was set up using low density
culture of fibroblasts that were in their Log phase characterized by
healthy and well-proliferating cells. In the case of HDFs, results of pro­
liferation assay were consistent with cytotoxicity test.
Fluorescent staining of the fibroblasts showed that surfaces of con­

trol, 2-LD-BG and 4-LD-BG were unfavorable to adhesion of BJ cells and
HDFs. There were single, spherical cells on the surface of mentioned
samples (Fig. 5a). Importantly, observed round cells were viable since
they exhibited green fluorescence. Surprisingly, 2-LD-AG material did
not allow for adhesion of BJ cells, but was favorable to adhesion of

HDFs. It should be noted that in the case of external wound dressing
applications, biomaterial surface that is unfavorable to skin cell adhe­
sion is very desired since it allows to remove or exchange the dressing
without disruption of the wound bed (Vivcharenko et al., 2021; Wojcik
et al., 2021). Performed live/dead staining of fibroblasts grown around
the tested samples showed their non-toxicity. Nevertheless, it should be
noted that ratio between culture medium and sample weight (1 ml of
medium per 30 mg sample) in direct contact test was different than the
one in cytotoxicity test (1 ml of medium per 100 mg sample). Materials
did not support cell adhesion but cells cultured around the samples were
healthy, viable, well-spread and with typical morphology (Fig. 5b).
Moreover no dead cells were detected around the samples. However,
there were noticeably fewer BJ cells around the 4-LD-BG material,
which is consistent with cytotoxicity test results.
Another model selected to evaluate the toxicity of synthesized
hydrogels was zebrafish. The zebrafish (Danio rerio) is known to show
high molecular, physiological, genetic and immunological similarity to
humans and is commonly accepted as a valid alternative to mammalian
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Carbohydrate Polymers 272 (2021) 118485

Fig. 5. CLSM images after live/dead staining (green fluorescence – viable cells, red fluorescence – dead cells) of fibroblasts cultured in the presence of tested samples:
(a) Fibroblast adhesion to the surface of tested samples (PS control – healthy and well attached cells grown on the surface of polystyrene well); (b) Viability of
fibroblasts cultured around the tested samples.

models for evaluation of toxicity and biocompatibility of novel bio­
materials. In our experiments, zebrafish embryos were maintained in
extracts collected from tested hydrogels obtained in the same mediumto-hydrogel proportion that in the tests of fibroblasts cytotoxicity. Ob­
servations made during 96 h post fertilization showed the lack of the
extracts influence on coagulation, tail detachment, development of so­
mites and heartbeat in the comparison with negative E3 control. Also,
there were no incidence and description of morphological and physio­
logical abnormalities of zebrafish during 4 days of incubation in the
extracts (Fig. 6a, b). Moreover, extracts of control, 2-LD-BG, 4-LD-BG
and 2-LD-AG curdlan hydrogels did not influence the locomotor activity
of 5 dpf zebrafish (one-way ANOVA: F(4,99) = 0.5283; p = 0.7152,
Fig. 6c). Thus, we may exclude the neurotoxic effects of the hydrogels.
Interestingly, the results of our experiments conducted on fibroblasts

(suggesting cytotoxicity of tested samples, at least in some of the per­
formed tests) and on zebrafish eggs and larvae (showing the complete
lack of toxicity and neurotoxicity of the samples) were not consistent.
However, it is known that results of biomaterials biological safety in
different toxicity models may vary significantly. For example, it was
observed that gypsum (calcium sulfate dihydrate), a popular bone void
filler, is highly cytotoxic in cell culture tests due to the quick dissolution
and release of significant amounts of calcium ions (Przekora et al., 2014)
but simultaneously it is claimed nontoxic in vivo and accepted for
commercial use (eg. 3i® Calcium Sulfate Bone Cement, Biomet Inc.,
USA; FDA approval in 2001). Therefore, slight cytotoxicity of poly(LDOPA)-modified curdlan (in particular, 2-LD-BG sample) is not a

contraindication to use the hydrogel as biomedical material because it
was simultaneously found safe in a sensitive zebrafish model.
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Fig. 6. Effects of control, and poly(L-DOPA) hydrogels on apical observation of acute toxicity (a), morphological changes at 96 hpf (hours post fertilization) zebrafish
larvae (b) and locomotor activity of 5 dpf (days post fertilization) zebrafish larvae (c).

As proved earlier (Burzio & Waite, 2000), catechol moieties of pol­
ycatecholamine coatings on different substrates allow to immobilize
different molecules with thiol and amine groups via Michael addition or
Schiff base reactions. We have confirmed this in our earlier report on
PDA-modified curdlan hydrogels coupled with gentamicin and peroxi­
dase (antibiotic and enzyme, respectively, with free amino group in their
structures) (Michalicha et al., 2021). Therefore, we decided to verify
whether poly(L-DOPA)-modified curdlan also binds gentamicin, with
positive impact on its antibacterial properties. Previous results of Kuang
et al. (2014) suggested that this strategy could be beneficial. Namely,
they observed that poly(L-DOPA) coating, formed in high ionic strength
environment, was thicker, allowed to attach/release more antibiotic
molecules and inhibited the bacterial growth more intensely than PDA
coating. According to the authors, this difference could be explained by
a significant presence of carboxyl groups preserved in poly(L-DOPA)
coatings formed in high salt buffer (Kuang et al., 2014). Therefore, we
performed pilot experiment to compare gentamicin binding to curdlan
hydrogels with and without pre-activation by EDC/NHS. EDC is a pop­

ular zero-length crosslinker known to react with carboxylic acid groups
of one compound, forming an amine-reactive O-acyl isourea interme­
diate which reacts with amine groups of another compound, yielding a
conjugate of the two molecules. The addition of NHS stabilizes the
amine-reactive intermediate, thus increasing the efficiency of EDCmediated coupling reactions. The results of the pilot experiment were
shown in Fig. 7. First, as expected, it was found that control pristine
curdlan did not bind any notable amount of gentamicin, due to the lack
of carboxyl groups and catechol moieties. Second, gentamicin was
bound to all poly(L-DOPA)-modified curdlan in amount 3300–4100 μg/g
of dry hydrogel weight. However, statistical but very small difference
between the amount of drug bound without and with EDC/NHS acti­
vation was observed only for 4-LD-BG sample. Surprisingly, effect of
EDC/NHS activation was not noted even for 2-LD-AG hydrogel, although
XPS tool allowed to detect free carboxyl groups in this sample. This
suggests that either carboxyl groups in poly(L-DOPA) deposits were
sterically unavailable for EDC coupling or the amount of free carboxyl
groups in tested hydrogels was too low to noticeably enhance the drug
bonding. Therefore, for further experiments we decided to bind genta­
micin to poly(L-DOPA)-modified curdlan simply by soaking in drug so­
lution and binding to poly(L-DOPA) catechol moieties, without EDC/

Fig. 7. Comparison of gentamicin immobilization (μg/g of dry hydrogel
weight) via binding by catechol moiety and via EDC/NHS activation of free
carboxyl groups; asterisk (*) indicate statistically significant results according
to Student’s t-test (p < 0.0002).

NHS activation.
In these experiments, performed on lyophilized hydrogel slices,
gentamicin was immobilized in amount of 3970–5479 μg/g dry hydro­
gel weight for poly(L-DOPA)-modified samples, in comparison with

861 μg/g dry hydrogel weight for control pristine curdlan. These
amounts were higher than those earlier observed for PDA-modified
curdlan hydrogel which ranged between 3164 and 3773 μg/g dry
hydrogel weight (Michalicha et al., 2021). Drug release profiles in closeloop system showed that gentamicin was partially released to PBS within
48–72 h: 83.7% of total drug for control curdlan, 50.57% – for 2-LD-BG,
44.08% – for 4-LD-BG and 53.76% – for 2-LD-AG (Fig. 8a,c). This cor­
responded to 720 μg gentamicin released by 1 g of dry weight of control
hydrogel, 2353 μg – for 2-LD-BG, 2415 μg – for 4-LD-BG and 2134 μg –
for 2-LD-AG (Fig. 8c). These amounts of gentamicin were released due to
a pore-dependent mechanism defined as Fickian diffusion, as suggested
by values of drug release exponent (n): 0.2532–0.2875 (Fig. 8c). The
remaining amount of gentamicin (16.3% for control and 46.2–55.4% for
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Carbohydrate Polymers 272 (2021) 118485

Fig. 8. Profiles of gentamicin release in: closed-loop system (a) and semi-open-loop system (b). Parameters of gentamicin release from curdlan hydrogel samples in
close-loop system (c).

poly(L-DOPA)-modified curdlan; Fig. 8c) was attached to the matrix via
more stable bonds, most probably mainly via catechol domains of poly
(L-DOPA). The results confirmed that poly(L-DOPA)-modified curdlan
released gentamicin much more efficiently than pristine curdlan, with
efficacy higher for 2-LD-BG and 4-LD-BG hydrogels than for 2-LD-AG
sample.
In semi-open-loop system, the drug release for control curdlan was
limited to 5 days while it was at least twice longer for all poly(L-DOPA)modified samples. Again, among the modified hydrogels, drug was

released the fastest for 2-LD-AG (Fig. 8b). Therefore, the sample modi­
fied by poly(L-DOPA) according to AG mode allowed to bind the less
antibiotic and released it the quickest in comparison to the samples
modified according to BG mode, suggesting that AG mode is less effec­
tive in drug binding as an alternative BG mode.
Evaluation of antibacterial activity according to AATCC 100–2004
standard for porous materials showed the similar and high antibacterial
potency of all poly(L-DOPA)-modified curdlan hydrogels against all
tested bacterial strains (100% of bacterial viability reduction; Fig. 9a).
However, the experiment in semi-open-loop system and related bacterial
adhesion revealed significant differences between three modified sam­
ples (Fig. 9b,c). Both samples obtained in BG mode were highly effective
in protection against Gram-positive bacterial strains (S. aureus and
S. epidermidis). These samples effectively inhibited bacterial growth for
up to 13 or 28 days (S. aureus) and 31 or 44 days (S. epidermidis) for 2LD-BG and 4-LD-BG sample, respectively (data were highlighted in red
font; Fig. 9b). Afterwards, bacterial growth appeared in treated extracts
but it did not reach the level of positive growth control until 47 days
(Fig. 9b) which suggested the drug release on very low sub-inhibitory
levels. In contrary, extracts of 2-LD-AG hydrogel inhibited the growth
of Gram-positive bacteria for only 4–9 days which is even worse that
results observed for control curdlan (8–9 days) (Fig. 9b). This is a

surprising observation because 2-LD-AG hydrogel contained high
amount of immobilized drug (2134 μg/g of dry hydrogel weight; Fig. 8c)
and drug release profile during first 8 days was quite similar to those of
2-LD-BG and 4-LD-BG samples (Fig. 8b). For Gram-negative E. coli strain,
extracts of poly(L-DOPA)-modified curdlan were protected against bac­
terial growth during significantly shorter period (which was limited to
maximum 1–8 days of complete protection and 12–13 days of partial
protection; Fig. 9b). The relations between antibacterial efficacy for all 4

tested samples were similar to those observed for Gram-positive strains –
2-LD-BG and 4-LD-BG hydrogels were more effective than control and 2LD-AG hydrogels.
Less striking differences between different poly(L-DOPA)-modified
samples were observed in relation to bacterial adhesion to hydrogels
subjected earlier to drug release in semi-open-loop system, as shown on
confocal microscopy images. All modified hydrogels were practically
free from adhered bacteria (with the exception of 2-LD-AG hydrogel
incubated with S. aureus), while abundant bacterial clusters were noted
for control curdlan (Fig. 9c).
Based on the profiles of drug release in semi-open-loop system and
antibacterial activity of samples extracts, it is likely that there are two
phases of gentamicin release from poly(L-DOPA)-modified curdlan
hydrogels. First, the intense drug burst appears during first days of the
release, producing high concentrations of antibiotic in the extracts.
Second, the remaining amount of drug is gradually released, yielding in
low (below 1 μg/ml) amount of antibiotic in the extracts which is close
to sub-inhibitory drug concentration. Moreover, it seems that BG
method is more efficient in gentamicin binding and antibacterial pro­
tection of curdlan hydrogel than AG method. What is important, we
compared the data obtained in this study with those noted for PDAmodified hydrogels tested in our previous report (Michalicha et al.,
2021). The amounts of immobilized gentamicin and period of full
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Carbohydrate Polymers 272 (2021) 118485

Fig. 9. Results of antibacterial activity test according to AATCC Test Method 100–2004 (a). Results of bacterial growth inhibition in semi-open-loop system pre­
sented as bacterial growth in extracts of curdlan hydrogels incubated with MH broth, exchanged daily and infected daily by bacterial inoculates (b). Confocal images

of curdlan hydrogels after the bacterial adhesion test (samples first subjected the drug release, then inoculated with bacterial suspension, washed and stained for live/
dead adhered bacteria). Green dots indicate the presence of live bacterial cells adhered to the matrix, as clusters (red arrows) and individual cells (yellow arrows) (c).

antibacterial
protection
for
poly(L-DOPA)-modified
curdlan
(3970–5479 μg/g dry hydrogel weight and 44 days, respectively) were
higher than those earlier observed for PDA-modified curdlan
(3164–3773 μg/g dry hydrogel weight and 28 days, respectively).
Therefore, we not only confirmed our hypothesis that levodopa may
effectively provide the biologically safe modification of polysaccharide
matrix which may be used for fabrication of wound dressings. It was also
found that poly(L-DOPA) modification may be even more efficient in
gentamicin binding and in the resulting antibacterial activity of such
modified curdlan hydrogel than PDA deposit.
An important conclusion can be withdrawn from he obtained results.
It concerns the consequences of AG and BG modes application for cur­
dlan hydrogel modification. It seems that poly(L-DOPA) deposition on
curdlan previously subjected to thermal gelation (AG mode) may lead to
concentration of polycatecholamine on curdlan microfibrils surface.
Thus, as shown for 2-LD-AG sample, poly(L-DOPA) can be more easily

detected on curdlan sample by XPS method, causes the increase of hy­
drophilicity of curdlan matrix (as expressed in an increased soaking
capacity), induces the proliferation of cells cultured with the sample,
favors the fibroblasts cells adhesion on the sample surface, lowers the
gentamicin binding and accelerates the drug release from the modified
hydrogel. On the other hand, L-DOPA monomer distribution in curdlan

suspension and poly(L-DOPA) polymerization inside the gelled curdlan
(BG mode) may lead to more uniform dispersion of polycatecholamine
within the entire curdlan microfibrils network. In such case, as shown
for 2-LD-BG sample, poly(L-DOPA) cannot be detected on curdlan sam­
ple by XPS method, sample shows similar soaking capacity as unmodi­
fied curdlan matrix, does not exhibit any beneficial effect on
proliferation or adhesion of fibroblasts cultured with the sample but
allows to bind more drug to the dispersed polycatecholamine and
consequently enables more sustained drug release. In relation to curdlan
hydrogel application as wound dressing, 2-LD-BG sample seems to be
13


Carbohydrate Polymers 272 (2021) 118485

A. Michalicha et al.

much more promising than 2-LD-AG curdlan hydrogel. In addition, the
increase of L-DOPA monomer concentration for the sample preparation
(from typically used 2 mg/ml to 4 mg/ml) results both in enhanced drug
immobilization and increased cytotoxicity of the hydrogel. Thus, this
strategy is less recommended in relation to wound dressing synthesis.

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4. Conclusions
The results obtained in this study lead to the conclusion that levo­
dopa may effectively modify the polysaccharide (curdlan) matrix and
may be used for fabrication of biologically safe wound dressings.
Moreover, the choice of levodopa instead of dopamine for subsequent
immobilization of free amine groups-containing drugs can be beneficial.
In some synthesis modes, poly(L-DOPA)-modified hydrogels were
nontoxic, in particular in zebrafish model, and unfavorable for fibro­
blasts adhesion which is important for the biomaterials designed as
wound dressings. Due to its clot-formation stimulating activity and
ability to bind and release antibiotics, they could be designed for healing
of hard-to-heal bleeding wounds endangered by infection. Thus, it seems
that levodopa deserves much more attention for nature-inspired modi­
fication of biomaterials than it attracts nowadays. However, it is
extremely important to carefully select the conditions for poly(L-DOPA)
deposition in the polymer matrix. Mode of this deposition may exert
very high impact on physicochemical and biological properties of suchproduced biomaterial.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2021.118485.
CRediT authorship contribution statement
Anna Michalicha: Investigation, Methodology, Formal analysis,
Writing – original draft. Agata Roguska: Investigation, Methodology,
Formal analysis, Writing – original draft. Agata Przekora: Investiga­

tion, Methodology, Formal analysis, Writing – original draft. Barbara
´ ska: Investigation, Formal analysis, Writing – original draft.
Budzyn
Anna Belcarz: Supervision, Conceptualization, Funding acquisition,
Methodology, Investigation, Formal analysis, Writing – original draft,
Project administration.
Acknowledgements
This study was financed from DS6/2021 grant (Medical University of
´jcik (Chair and
Lublin, Poland). Authors are grateful to Michał Wo
Department of Biochemistry and Biotechnology, Medical University of
Lublin, Poland) for his help in performance of confocal and DIC mi­
croscopy and to Prof. Marcin Pisarek (Institute of Physical Chemistry,
Polish Academy of Sciences, Poland) for help with XPS measurements.
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